Control Of Glycogen Metabolism Research Articles

A Homozygous PPP1R21 Splice Variant Associated with Severe Developmental Delay, Absence of Speech, and Muscle Weakness Leads to Activated Proteasome Function

PPP1R21 acts as a co-factor for protein phosphatase 1 (PP1), an important serine/threonine phosphatase known to be essential for cell division, control of glycogen metabolism, protein synthesis, and muscle contractility. Bi-allelic pathogenic variants in PPP1R21 were linked to a neurodevelopmental disorder with hypotonia, facial dysmorphism, and brain abnormalities (NEDHFBA) with pediatric onset. Functional studies unraveled impaired vesicular transport as being part of PPP1R21-related pathomechanism. To decipher further the pathophysiological processes leading to the clinical manifestation of NEDHFBA, we investigated the proteomic signature of fibroblasts derived from the first NEDHFBA patient harboring a splice-site mutation in PPP1R21 and presenting with a milder phenotype. Proteomic findings and further functional studies demonstrate a profound activation of the ubiquitin–proteasome system with presence of protein aggregates and impact on cellular fitness and moreover suggest a cross-link between activation of the proteolytic system and cytoskeletal architecture (including filopodia) as exemplified on paradigmatic proteins including actin, thus extending the pathophysiological spectrum of the disease. In addition, the proteomic signature of PPP1R21-mutant fibroblasts displayed a dysregulation of a variety of proteins of neurological relevance. This includes increase proteins which might act toward antagonization of cellular stress burden in terms of pro-survival, a molecular finding which might accord with the presentation of a milder phenotype of our NEDHFBA patient.

719-P: Inducible Estrogen-Related Receptor Alpha Overexpression Stimulates Skeletal Muscle Oxidative Metabolism and Improves Exercise Endurance in Mice

Aerobic exercise promotes skeletal muscle insulin sensitivity by enhancing glucose uptake and storage and by increasing mitochondrial oxidative capacity. Exercise-responsive transcriptional pathways that regulate metabolism represent potential therapeutic targets to combat insulin resistance. The ERRα nuclear receptor regulates mitochondrial oxidative metabolism and is required for the skeletal muscle metabolic response to exercise. In the current study investigated whether ERRα expression is sufficient to drive exercise-induced adaptations in muscle. We generated conditional ERRα transgenic mice that overexpress human ERRα in a tetracycline-inducible and skeletal muscle-specific manner (ERRαMOEiHSA). Dox-induced ERRαMOEiHSA (versus non-induced) mice exhibited reddening in glycolytic muscles, a characteristic of oxidative muscle. Mitochondrial content was increased in these muscles, based on mtDNA quantitation and citrate synthase activity. Consistently, ERRα-overexpressing muscles had higher O2 consumption rates and increased glycogen content. To determine if these metabolic changes altered exercise capacity, run performance was assessed in untrained mice. Dox-induced ERRαMOEiHSA mice tested at constant speed (endurance test) or at increasing speeds (aerobic test) ran longer compared to controls. Post-run blood glucose levels were higher in both testing paradigms, which may be due to greater reliance on β-oxidation and higher basal glycogen stores in ERRα-expressing muscles. Using ChIP and reporters assays we showed that ERRα directly activates gene encoding the catalytic (Ppp1ca) and regulatory subunits (Ppp1r3c) of protein phosphatase 1, involved in hormonal and contractile-activity control of glycogen metabolism. Collectively, these results demonstrate that short-term ERRα activation promotes muscle metabolic adaptations and exercise performance similar to exercise training. Disclosure J. Li: None. A. Hamilton: None. J.M. Huss: None. Funding American Diabetes Association (1-18-IBS-103 to J.M.H.)

Abstract 2926: The glycogen synthase kinase-3 inhibitor, 9-ING-41, synergizes with chemotherapy to inhibit pancreatic tumor growth in vivo

Abstract Glycogen synthase kinase-3 (GSK3) is a ubiquitously expressed serine-threonine protein kinase involved in multiple cellular functions ranging from the control of glycogen metabolism to transcriptional regulation. We have previously demonstrated that GSK-3β is overexpressed in human pancreatic ductal adenocarcinoma (PDAC) and aberrant nuclear accumulation of GSK-3β serves as a hallmark of poorly differentiated PDAC. Moreover, genetic ablation of GSK-3β or GSK-3 inhibition led to PDAC tumor cell killing, identifying GSK-3β as a therapeutic target in PDAC. Herein, we describe a novel GSK-3 inhibitor, 9-ING-41. The effect of 9-ING-41 on tumor cell growth was tested using several PDAC and patient-derived xenograft (PDX) cell lines in vitro and in vivo using an orthotopic pancreatic cancer animal model. Interestingly, 9-ING-41 treatment resulted in the stabilization of cyclin B levels as well as enhanced serine-10 phosphorylated histone H3 (pS10HH3) and consequently M phase arrest and blocked mitotic exit. The prolonged M phase arrest induced mitotic catastrophe in pancreatic cancer cell lines and inhibited in vitro colony formation. An in vivo orthotopic pancreatic cancer animal model study using PDX-derived cell lines demonstrated that 9-ING-41 hindered tumor growth in combination with several standard chemotherapeutic agents. Significantly, we found that 9-ING-41 treatment impaired the DNA damage response pathway activated by Gemcitabine in pancreatic cancer cell lines. In conclusion, our study indicates that GSK-3 plays an important role in pancreatic cancer progression and 9-ING-41 in combination with existing gemcitabine or irinotecan could be a new strategy for treating patients with PDAC. Citation Format: Li Ding, Jin-san Zhang, John R. Dube, Vijay S. Madamsetty, Daniel M. Schmitt, Debabrata Mukhopadhyay, Daniel D. Billadeau. The glycogen synthase kinase-3 inhibitor, 9-ING-41, synergizes with chemotherapy to inhibit pancreatic tumor growth in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2926.

Amaryllidaceae Alkaloids as Potential Glycogen Synthase Kinase-3β Inhibitors.

Glycogen synthase kinase-3β (GSK-3β) is a multifunctional serine/threonine protein kinase that was originally identified as an enzyme involved in the control of glycogen metabolism. It plays a key role in diverse physiological processes including metabolism, the cell cycle, and gene expression by regulating a wide variety of well-known substances like glycogen synthase, tau-protein, and β-catenin. Recent studies have identified GSK-3β as a potential therapeutic target in Alzheimer´s disease, bipolar disorder, stroke, more than 15 types of cancer, and diabetes. GSK-3β is one of the most attractive targets for medicinal chemists in the discovery, design, and synthesis of new selective potent inhibitors. In the current study, twenty-eight Amaryllidaceae alkaloids of various structural types were studied for their potency to inhibit GSK-3β. Promising results have been demonstrated by alkaloids of the homolycorine-{9-O-demethylhomolycorine (IC50 = 30.00 ± 0.71 µM), masonine (IC50 = 27.81 ± 0.01 μM)}, and lycorine-types {caranine (IC50 = 30.75 ± 0.04 μM)}.

GNIP1 E3 ubiquitin ligase is a novel player in regulating glycogen metabolism in skeletal muscle

BackgroundGlycogenin-interacting protein 1 (GNIP1) is a tripartite motif (TRIM) protein with E3 ubiquitin ligase activity that interacts with glycogenin. These data suggest that GNIP1 could play a major role in the control of glycogen metabolism. However, direct evidence based on functional analysis remains to be obtained. ObjectivesThe aim of this study was 1) to define the expression pattern of glycogenin-interacting protein/Tripartite motif containing protein 7 (GNIP/TRIM7) isoforms in humans, 2) to test their ubiquitin E3 ligase activity, and 3) to analyze the functional effects of GNIP1 on muscle glucose/glycogen metabolism both in human cultured cells and in vivo in mice. ResultsWe show that GNIP1 was the most abundant GNIP/TRIM7 isoform in human skeletal muscle, whereas in cardiac muscle only TRIM7 was expressed. GNIP1 and TRIM7 had autoubiquitination activity in vitro and were localized in the Golgi apparatus and cytosol respectively in LHCN-M2 myoblasts. GNIP1 overexpression increased glucose uptake in LHCN-M2 myotubes. Overexpression of GNIP1 in mouse muscle in vivo increased glycogen content, glycogen synthase (GS) activity and phospho-GSK-3α/β (Ser21/9) and phospho-Akt (Ser473) content, whereas decreased GS phosphorylation in Ser640. These modifications led to decreased blood glucose levels, lactate levels and body weight, without changing whole-body insulin or glucose tolerance in mouse. ConclusionGNIP1 is an ubiquitin ligase with a markedly glycogenic effect in skeletal muscle.

A protein kinase screen of Neurospora crassa mutant strains reveals that the SNF1 protein kinase promotes glycogen synthase phosphorylation.

Glycogen functions as a carbohydrate reserve in a variety of organisms and its metabolism is highly regulated. The activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of the synthesis and degradation processes, respectively, are regulated by allosteric modulation and reversible phosphorylation. To identify the protein kinases affecting glycogen metabolism in Neurospora crassa, we performed a screen of 84 serine/threonine kinase knockout strains. We identified multiple kinases that have already been described as controlling glycogen metabolism in different organisms, such as NcSNF1, NcPHO85, NcGSK3, NcPKA, PSK2 homologue and NcATG1. In addition, many hypothetical kinases have been implicated in the control of glycogen metabolism. Two kinases, NcIME-2 and NcNIMA, already functionally characterized but with no functions related to glycogen metabolism regulation, were also identified. Among the kinases identified, it is important to mention the role of NcSNF1. We showed in the present study that this kinase was implicated in glycogen synthase phosphorylation, as demonstrated by the higher levels of glycogen accumulated during growth, along with a higher glycogen synthase (GSN) ±glucose 6-phosphate activity ratio and a lesser set of phosphorylated GSN isoforms in strain Ncsnf1KO, when compared with the wild-type strain. The results led us to conclude that, in N. crassa, this kinase promotes phosphorylation of glycogen synthase either directly or indirectly, which is the opposite of what is described for Saccharomyces cerevisiae. The kinases also play a role in gene expression regulation, in that gdn, the gene encoding the debranching enzyme, was down-regulated by the proteins identified in the screen. Some kinases affected growth and development, suggesting a connection linking glycogen metabolism with cell growth and development.

Neurodegeneration and functional impairments associated with glycogen synthase accumulation in a mouse model of Lafora disease

Lafora disease (LD) is caused by mutations in either the laforin or malin gene. The hallmark of the disease is the accumulation of polyglucosan inclusions called Lafora Bodies (LBs). Malin knockout (KO) mice present polyglucosan accumulations in several brain areas, as do patients of LD. These structures are abundant in the cerebellum and hippocampus. Here, we report a large increase in glycogen synthase (GS) in these mice, in which the enzyme accumulates in LBs. Our study focused on the hippocampus where, under physiological conditions, astrocytes and parvalbumin-positive (PV+) interneurons expressed GS and malin. Although LBs have been described only in neurons, we found this polyglucosan accumulation in the astrocytes of the KO mice. They also had LBs in the soma and some processes of PV+ interneurons. This phenomenon was accompanied by the progressive loss of these neuronal cells and, importantly, neurophysiological alterations potentially related to impairment of hippocampal function. Our results emphasize the relevance of the laforin–malin complex in the control of glycogen metabolism and highlight altered glycogen accumulation as a key contributor to neurodegeneration in LD.

Physiological Control of Liver Glycogen Metabolism: Lessons from Novel Glycogen Phosphorylase Inhibitors

Liver glycogen is synthesized in the postprandial state in response to elevated concentrations of glucose and insulin or by activation of neuroendocrine signals and it is degraded in the postabsorptive state in response to changes in the concentrations of insulin and counter-regulatory hormones. Dysregulation of either glycogen degradation or synthesis through changes in allosteric control or covalent modification of glycogen phosphorylase and glycogen synthase leads to disturbance of blood glucose homeostasis. Liver glycogen phosphorylase has a dual role in the control of glycogen metabolism by regulation of both glycogen degradation and synthesis. The phosphorylated form (GPa) is the active form and determines the rate of degradation of glycogen and it is also a potent allosteric inhibitor of the protein complex, involving the glycogen targeting protein G(L) and protein phosphatase-1, which catalyses dephosphorylation (activation) of glycogen synthase. Drug discovery programmes exploring the validity of glycogen phosphorylase as a therapeutic target for type 2 diabetes have generated a wide array of selective phosphorylase ligands that modulate the catalytic activity and / or the phosphorylation state (interconversion of GPa and GPb) as well as the binding of GPa to the allosteric site of G(L). Glycogen phosphorylase inhibitors that act in hepatocytes either exclusively by dephosphorylating GPa (e.g. indole carboxamides) or by allosteric inhibition of GPa (1,4-dideoxy-1,4-D-arabinitol) are very powerful experimental tools to determine the relative roles of covalent modification of glycogen phosphorylase and/or cycling between glycogen synthesis and degradation in the mechanism(s) by which insulin and neurotransmitters regulate hepatic glycogen metabolism.

Regulation of glycogen synthesis by the laforin–malin complex is modulated by the AMP-activated protein kinase pathway

Lafora progressive myoclonus epilepsy (LD) is a fatal autosomal recessive neurodegenerative disorder characterized by the presence of glycogen-like intracellular inclusions called Lafora bodies. LD is caused by mutations in two genes, EPM2A and EPM2B, encoding respectively laforin, a dual-specificity protein phosphatase, and malin, an E3 ubiquitin ligase. Previously, we and others have suggested that the interactions between laforin and PTG (a regulatory subunit of type 1 protein phosphatase) and between laforin and malin are critical in the pathogenesis of LD. Here, we show that the laforin-malin complex downregulates PTG-induced glycogen synthesis in FTO2B hepatoma cells through a mechanism involving ubiquitination and degradation of PTG. Furthermore, we demonstrate that the interaction between laforin and malin is a regulated process that is modulated by the AMP-activated protein kinase (AMPK). These findings provide further insights into the critical role of the laforin-malin complex in the control of glycogen metabolism and unravel a novel link between the energy sensor AMPK and glycogen metabolism. These data advance our understanding of the functional role of laforin and malin, which hopefully will facilitate the development of appropriate LD therapies.

Phosphatase as Starch Control

Plant metabolism shifts dramatically with the daily light cycle, from a situation in which carbon is fixed into sugars and stored as starch during photosynthesis to one in which reserves of starch are used for energy during the night. Precisely how this switch from biosynthesis to consumption is regulated has not been clear. But now Sokolov et al. have characterized an enzyme that may couple metabolism of starch to daily cycles. Taking a cue from the knowledge that in animals, accumulation of glycogen is regulated by a dual-specificity phosphatase (that is, one that modifies both phosphotyrosine and phosphoserine or phosphothreonine residues), the authors characterized a related phosphatase that they call DSP4. Mutant Arabidopsis plants in which the DSP4 gene was altered showed increased accumulation of starch in the leaves. DSP4 also interacted directly with starch granules in in vitro binding assays and, in plants expressing DSP4 tagged with green fluorescent protein, DSP4 was associated with starch granules during the day but not at night. The pH of the chloroplast stroma increases from about pH7 at night to about pH8 in the daytime. The redox environment also changes, with various enzymes being reduced during the day. The authors propose that these signals may couple to control of starch metabolism through DSP4 because DSP4 activity in in vitro assays was lost as pH increased from pH7 to pH8. Addition of oxidants decreased binding of DSP4 to starch granules and decreased the activity of the phosphatase. Thus, plants may have a phosphorylation-mediated mechanism controlling storage of energy in starch that resembles that of animals for control of glycogen metabolism. A related paper recently appeared in the Journal of Biological Chemistry . L. N. Sokolov, J. R. Dominguez-Solis, A.-L. Allary, B. B. Buchanan, S. Luan, A redox-regulated chloroplast protein phosphatase binds to starch diurnally and functions in its accumulation. Proc. Natl. Acad. Sci. U.S.A. 103 , 9732-9737 (2006). [Abstract] [Full Text] T. Niittylä, S. Comparot-Moss, W.-L. Lue, G. Messerli, M. Trevisan, M. D. J. Seymour, J. A. Gatehouse, D. Villadsen, S. M. Smith, J. Chen, S. C. Zeeman, A. M. Smith, Similar protein phosphatases control starch metabolism in plants and glycogen metabolism in mammals. J. Biol. Chem. 281 , 11815-11818 (2006). [Abstract] [Full Text]

GSK3 inhibitors: development and therapeutic potential.

Glycogen synthase kinase-3 (GSK3) was initially identified more than two decades ago as an enzyme involved in the control of glycogen metabolism. In recent years it has been shown to have key roles in regulating a diverse range of cellular functions, which have prompted efforts to develop GSK3 inhibitors as therapeutics. Here, we describe the biology of GSK3 relevant to its potential as a target for diabetes and neurodegenerative diseases, and discuss progress in the development of GSK3 inhibitors.

Three-Dimensional Structure of Phosphorylase Kinase at 22 Å Resolution and Its Complex with Glycogen Phosphorylase b

Phosphorylase kinase (PhK) integrates hormonal and neuronal signals and is a key enzyme in the control of glycogen metabolism. PhK is one of the largest of the protein kinases and is composed of four types of subunit, with stoichiometry (αβγδ) 4 and a total MW of 1.3 × 10 6. PhK catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a (GPa) and the stimulation of glycogenolysis. We have determined the three-dimensional structure of PhK at 22 Å resolution by electron microscopy with the random conical tilt method. We have also determined the structure of PhK decorated with GPb at 28 Å resolution. GPb is bound toward the ends of each of the lobes with an apparent stoichiometry of four GPb dimers per (αβγδ) 4 PhK. The PhK/GPb model provides an explanation for the formation of hybrid GPab intermediates in the PhK-catalyzed phosphorylation of GPb.

A novel protein tyrosine phosphatase gene is mutated in progressive myoclonus epilepsy of the Lafora type (EPM2).

Progressive myoclonus epilepsy of the Lafora type or Lafora disease (EPM2; McKusick no. 254780) is an autosomal recessive disorder characterized by epilepsy, myoclonus, progressive neurological deterioration and glycogen-like intracellular inclusion bodies (Lafora bodies). A gene for EPM2 previously has been mapped to chromosome 6q23-q25 using linkage analysis and homozygosity mapping. Here we report the positional cloning of the 6q EPM2 gene. A microdeletion within the EPM2 critical region, present inhomozygosis in an affected individual, was found to disrupt a novel gene encoding a putative protein tyrosine phosphatase (PTPase). The gene, denoted EPM2, presents alternative splicing in the 5' and 3' end regions. Mutational analysis revealed that EPM2 patients are homozygous for loss-of-function mutations in EPM2. These findings suggest that Lafora disease results from the mutational inactivation of a PTPase activity that may be important in the control of glycogen metabolism.

Expression of the gene encoding glycogen phosphorylase is elevated in diabetic rat skeletal muscle and is regulated by insulin and cyclic AMP.

Glycogen phosphorylase regulates the breakdown of glycogen into glucose, but as previous studies have demonstrated, the control of glycogen metabolism becomes deregulated in diabetes mellitus. Messenger RNA levels encoding several different proteins are altered in skeletal muscle biopsies of patients with insulin-dependent and non-insulin-dependent diabetes. The possible alteration of expression of the gene encoding the skeletal muscle isoform of glycogen phosphorylase during diabetes has not previously been investigated. We examined the effect of streptozotocin-induced diabetes and insulin treatment on glycogen phosphorylase mRNA in rat skeletal muscle; glycogen phosphorylase mRNA levels were elevated in diabetic rat muscle tissue, but were partially suppressed in diabetic rat muscle following insulin treatment. To distinguish between the effects of insulin and counter-regulatory hormones on glycogen phosphorylase mRNA levels, we employed differentiating rat L6 myoblasts in culture. Insulin stimulated the accumulation of glycogen phosphorylase mRNA as determined by Northern blot analysis. Moreover, insulin and dibutyryl cAMP stimulated expression of a transiently transfected chloramphenicol acetyl transferase reporter gene under the control of the muscle glycogen phosphorylase promoter in differentiating myotubes in culture, suggesting that the effects of insulin and counter-regulatory hormones on glycogen phosphorylase mRNA are at the level of transcription. These results suggest that insulin and epinephrine may participate in the induction of the glycogen phosphorylase gene during myogenesis; moreover, activation of this gene in muscle tissue may be a contributing factor in impaired glycogen storage during uncontrolled diabetes.

Role of Protein Phosphatase 2A in the Control of Glycogen Metabolism in Yeast

The yeast homologues of mammalian protein phosphatase 2A (PP2A) are encoded by two genes, PPH21 and PPH22. To evaluate the role of these phosphatases in the control of glycogen metabolism, wild-type cells and mutants carrying deletions of PPH21 or PPH22 were studied. Our results indicate that the lack of a single gene product does not result in significant changes in glycogen content, glycogen synthase, and glycogen phosphorylase activities. Since the double disruption is very detrimental to the cell, the effect of lack of PP2A was evaluated by using strain H336, which carries a deletion of the PPH21 gene and has the PPH22 gene placed under the control of the GAL1 promoter, under conditions that allowed either progressive depletion or overexpression of PPH22. When grown on galactose, H336 cells contain 2-3-fold more PP2A activity than control cells. After 14 h in glucose, however, PP2A activity in strain H336 is markedly reduced. The decrease in PP2A activity correlates with a reduced accumulation of glycogen and a more pronounced inactivation of glycogen synthase while glycogen phosphorylase becomes more resistant to inactivation. These observations suggest a role for PP2A in controlling the activation states of both enzymes. The total amount of phosphorylase was also higher in the PP2A-depleted cells, as determined by both enzymic and immunochemical techniques. However, Northern-blot analysis revealed that this is not due to an increase in the phosphorylase mRNA, which is in fact reduced in these cells. In contrast, overexpression of PP2A causes an increased expression of glycogen phosphorylase and a resulting failure to accumulate glycogen. We conclude that PP2A is involved in regulating both the amounts and the activation states of glycogen synthase and glycogen phosphorylase.

The Saccharomyces SHP1 Gene, Which Encodes a Regulator of Phosphoprotein Phosphatase 1 with Differential Effects on Glycogen Metabolism, Meiotic Differentiation, and Mitotic Cell Cycle Progression

The phosphoprotein phosphatase 1 (PP1) catalytic subunit encoded by the Saccharomyces GLC7 gene is involved in control of glycogen metabolism, meiosis, translation, chromosome segregation, cell polarity, and G2/M cell cycle progression. It is also lethal when overproduced. We have isolated strains which are resistant to Glc7p overproduction lethality as a result of mutations in the SHP1 (suppressor of high-copy PP1) gene, which was previously encountered in a genomic sequencing project as an open reading frame whose interruption totally blocked sporulation and slightly slowed cell proliferation. These phenotypes also characterized our shp1 mutations, as did deficient glycogen accumulation. Lysates from the shp1 mutants were deficient in PP1 catalytic activity but exhibited no obvious abnormalities in the steady-state level or subcellular localization pattern of a catalytically active Glc7p-hemagglutinin fusion polypeptide. The lower level of PP1 activity in shp1 cells permitted substitution of a galactose-induced GAL10-GLC7 fusion for GLC7; depletion of Glc7p from these cells by growth in glucose medium resulted in G2/M arrest as previously observed for a glc7cs allele but with depletion arrest occurring most frequently at a later stage of mitosis. The higher requirement of glycogen accumulation and sporulation for PP1 activity would permit their regulation via Glc7p activity, independent of its requirement for mitosis.

Role of Protein Phosphatase 2A in the Control of Glycogen Metabolism in Yeast

Role of Protein Phosphatase 2A in the Control of Glycogen Metabolism in Yeast

Cell swelling and the control of glycogen metabolism in rat hepatocytes

Cell swelling and the control of glycogen metabolism in rat hepatocytes

Control of glycogen metabolism by gluconeogenic and ketogenic substrates in isolated hepatocytes from fed rats

1. 1. This study was conducted to examine the effects of gluconeogenic and ketogenic substrates on the activities of the glycogen-metabolizing enzymes and on glycogenolysis in isolated hepatocytes from fed rats. 2. 2. Gluconeogenic substrates like fructose, dihydroxyacetone or lactate turned out to stimulate the glucose-induced activation of glycogen synthase and this effect may be linked, to some extent, to the increase of the cellular glucose 6-phosphate concentration. 3. 3. The effect of fructose was accompanied by the onset of glycogen synthesis. 4. 4. Energetic substrates like fatty acids were also potent activators of glycogen synthase, especially in the presence of glucose. 5. 5. When fatty acids were added alone or together with a physiological concentration of glucose, they induced or potentiated the inhibition of glycogen phosphorylase-a. 6. 6. This inhibitory effect was mediated by a decrease of lactate release. 7. 7. The stimulatory effect of amino acids on glycogen synthase seemed to be direct, non mediated by an inhibition of the phosphorylase-a activity although hepatic glycogenolysis markedly decreased. 8. 8. Moreover, the amino acid action could be linked to their capacities to induce cell swelling and/or to limit proteolysis.

Molecular cloning and expression of the regulatory (RG1) subunit of the glycogen-associated protein phosphatase

DNA clones encoding the glycogen-binding (RG1) subunit of glycogen-associated protein phosphatase were isolated from rabbit skeletal muscle lambda gt11 cDNA libraries. Overlapping clones provided an open reading frame of 3327 nucleotides that predicts a polypeptide of 1109 amino acids with a molecular weight of 124,257. Northern hybridization of rabbit RNA identified a major mRNA transcript of 7.5 kilobases present in skeletal, diaphragm, and cardiac muscle, but not in brain, kidney, liver, and lung. Southern analysis of rabbit genomic DNA digested with various restriction endonucleases gave rise to a single hybridizing fragment, suggesting that a single gene is present. Expression of the complete RG1 subunit coding sequence in Escherichia coli generated a protein of apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 160,000, similar to the size of the polypeptide detected by Western immunoblot in rabbit skeletal muscle extracts. The RG1 subunit shares significant homology with the Saccharomyces cerevisiae GAC1 gene product which is involved in activation of glycogen synthase and glycogen accumulation. The homology with GAC1 substantiates the role of this enzyme in control of glycogen metabolism. Hydropathy analysis of the RG1 subunit amino acid sequence revealed the presence of a hydrophobic region in the COOH terminus, suggesting a potential association with membrane. This result suggests that the same phosphatase regulatory component may be involved in targeting the enzyme both to membranes and to glycogen.